Cocaine and heroin are metabolized by carboxylesterases (CE) present in the liver. With cocaine, the methyl group is removed by human carboxylesterase 1 (hCE1) to produce benzyl ecgonine, the major inactive metabolite of the drug. With heroin, both methyl esters are sequentially hydrolyzed by the same enzyme to yield 6-monoacetylmorphine and subsequently, morphine. We have recently determined the x-ray crystal structure of hCE1, in complex with the cocaine and heroin analogs, homatropine and naloxone, respectively. Using this information, we will engineer mutations within the hCE1 amino acid sequence to improve its ability to hydrolyze cocaine. These enzymes will be tested in an esterase-deficient animal model to determine whether they can be used to purge cocaine from the blood stream. Additionally, we will determine the ability of these mutants to transesterifiy cocaine in the presence of ethanol to form cocaethylene, and measure the hydrolysis of this compound by different hCE1 proteins. [unreadable] [unreadable] The Specific Aims of this application are therefore to: 1) generate hCE1 mutants with improved catalytic efficiency for cocaine hydrolysis; 2) determine the amino acid residues involved in the transesterification reaction of cocaine to cocaethylene by hCE1; 3) elucidate crystal structures of engineered forms of hCE1 in complexes with cocaine and cocaine analogues; and 4) assess the impact of mutant forms of hCE1 on plasma esterase-deficient mice exposed to cocaine. Overall, these studies should allow development of novel therapeutic approaches for the treatment of cocaine overdose. [unreadable] [unreadable]